Brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 activate human ADP- ribosylation factors ARF1 and ARF3 by catalyzing the replacement of ARF-bound GDP with GTP to regulate Golgi vesicular transport. We showed this year a requirement for BIG1 (but not BIG2) in glycosylation and function of integrin beta1. In cells treated for two or three days with BIG1, but not BIG2, siRNA, both amounts and electrophoretic mobility of the initially 130-kDa beta1 were increased. Peptide N-glycosidase F digestion converted all beta1 to an 85-kDa (core protein) form. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa beta1 was not seen. By electron microscopy, Golgi membranes in BIG1-depleted cells were less sharply defined than those in mock or BIG2 siRNA-treated cells, with more vesicle-like structures at the trans face. Amounts of active RhoA-GTP also were decreased in such cells and were restored after overexpression of HA-BIG1. Aberrant beta1 was present on the cell surface, but its function in cell spreading, adhesion, and migration was impaired. By immunofluorescence microscopy, BIG1 siRNA-treated cells showed less spreading and concentration of beta1 at the surface than those treated with BIG2 or control siRNA. These results indicate a previously unrecognized requirement for BIG1 in the glycosylation of beta1 by Golgi enzymes, which is critical for integrin function in developmental and other vital cell processes.[unreadable] [unreadable] We had earlier identified sites for binding protein kinase A, i.e., A kinase-anchoring protein (AKAP) sequences, in both BIG1 and BIG2 molecules. More recently, we showed that elevation of cell cAMP content caused PKA-catalyzed phosphorylation and nuclear accumulation of BIG1 but not BIG2. The next question was whether the phosphorylation altered GEP activity. Incubation of BIG1 or BIG2 with PKA catalytic subunit and ATP resulted in retardation of its electrophoretic migration, consistent with PKA phosphorylation. Okadaic acid inhibits many protein phosphatases, including protein phosphatase 1 (PP1) and PP2A, which can reverse PKA-catalyzed phosphorylation. Incubation of cells with okadaic acid caused concentration-dependent accumulation of presumably phosphorylated BIG1 and BIG2 with decreased mobility, which was increased by subsequent incubation in vitro with specific recombinant phosphatases, PP1gamma>PP2A>>PP1alpha. To assay GEP activity, BIG1 and BIG2 were immunoprecipitated from cells that had been depleted, respectively, of BIG2 or BIG1 by using specific siRNA. GEP activity of each was significantly decreased after incubation with recombinant PKA plus ATP and restored by incubation with PP1gamma. Consistent with a role for PP1gamma in regulation of BIG phosphorylation, endogenous PP1gamma, but not PP1alpha or beta, was immunoprecipitated with BIG1 or BIG2 from microsomal fractions. All of our data are consistent with roles for BIG1 and BIG2 phosphorylation in vesicular trafficking via alterations in ARF activation, and regulatory functions for cAMP, PKA, and PP1gamma in ARF activation by BIG1 and BIG2. Identification of the specific PKA regulatory R subunits and AKAP sequences in BIG molecules that are involved is continuing.[unreadable] [unreadable] The type 1, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: cytokine-inducible proteolytic cleavage of TNFR1 ectodomains and constitutive release of full-length TNFR1 in exosome-like vesicles. Dr. Aminul Islam and Dr. Stewart Levine had found that regulation of both types of TNFR1 release appeared to involve intracellular trafficking of TNFR1 cytoplasmic vesicles. We then collaborated to determine whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of depletion of endogenous proteins with specific RNA interference (siRNA) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and TNFR1 was co-immunoprecipitated with BIG2. Association between BIG2 and TNFR1 was disrupted by BFA, which inhibits the ARF activation by BIG1 and BIG2 that is required for vesicular trafficking. Consistent with the preferential activation of class 1 ARFs by BIG2, it was shown that ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion as determined by specific siRNA depletion of individual ARFs. We conclude that an association between BIG2 and TNFR1 selectively regulates extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.[unreadable] [unreadable] Protein ubiquitinylation, which has key roles in several important cellular processes, requires the E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and usually, a substrate-specific E3 ubiquitin-protein ligase. In one class of E3 ubiquitin ligases, the catalytic domain contains a zinc-binding RING finger motif. ARD1 (ADP-ribosylation factor domain protein 1)initially cloned in our laboratory because of its C-terminal ARF domain, contains a RING finger structure near the N-terminus followed by two predicted B-Boxes and a coiled-coil protein-interaction motif. This makes it a member of the TRIM (Tripartite motif) or RBCC (RING, B-Box, coiled-coil) family. The region containing B-Boxes and coiled-coil sequence serves also as a GTPase-activating protein for the ADP-ribosylation factor domain of ARD1. We demonstrated that full-length ARD1 or the RING finger domain (residues 1-110) produced polyubiquitinylated proteins in vitro when incubated with purified mammalian E1 and specific recombinant E2 enzymes plus ATP, and ubiquitin. Deletion of the RING region or point mutations within the RING sequence abolished ARD1 E3 ligase activity. All data were consistent with a potential function for ARD1 as an E3 ubiquitin ligase in cells.